This application claims the benefit of priority to Germany 101 06 459.4 filed Feb. 13, 2001.
1. Field of the Invention
The present invention relates to a process for the fermentive preparation of D-pantothenic acid and/or its salts or mixtures containing these using microorganisms from the Enterobacteriaceae family, in which at least one or more genes from the group gcvT, gcvH and gcvP are enhanced.
2. Discussion of the Background
Pantothenic acid is produced all over the world in amounts of several thousand tons per year. It is used, inter alia, in human medicine, in the pharmaceutical industry, and in the foodstuffs industry. A high proportion of the pantothenic acid produced is used for feeding economically useful animals such as poultry and pigs. The demand for this material is increasing.
Pantothenic acid can be prepared by chemical synthesis or biotechnically by the fermentation of suitable microorganisms in suitable nutrient media. In the case of chemical synthesis, DL-pantolactone is an important precursor. This compound is prepared in a multi-step process from formaldehyde, isobutylaldehyde and cyanide, the racemic mixture is resolved in a subsequent process step, D-pantolactone is condensed with xcex2-alanine, and D-pantothenic acid is obtained in this way.
The typical commercial form is the calcium salt of D-pantothenic acid. The calcium salt of the racemic mixture D,L-pantothenic acid is also commonly available.
The advantage of fermentative preparation by microorganisms is the direct formation of the desired stereoisomeric form, that is the D-form, which contains no L-pantothenic acid.
Various species of bacteria such as, e.g. Escherichia coli (E. coli), Arthrobacter ureafaciens, Corynebacterium erythrogenes, Brevibacterium ammoniagenes and also yeasts, such as, e.g. Debaromyces castellii can, as shown in EP-A 0 493 060, produce D-pantothenic acid in a nutrient medium which contains glucose, DL-pantoic acid and xcex2-alanine. Furthermore, EP-A 0 493 060 shows that, in the case of E. coli, the formation of D-pantothenic acid is improved by the amplification of pantothenic acid biosynthesis genes from E. coli which are contained on the plasmids pFV3 and pFV5, in a nutrient medium which contains glucose, DL-pantoic acid and xcex2-alanine.
EP-A 0 590 857 and U.S. Pat. No. 5,518,906 describe mutants derived from E. coli strain IF03547, such as FV5714, FV525, FV814, FV521, FV221, FV6051 and FV5069 which carry resistance to various antimetabolites such as salicylic acid, xcex1-ketobutyric acid, xcex2-hydrocyaspartic acid, O-methylthreonine and xcex1-ketoisovaleric acid. They produce pantoic acid in a nutrient medium which contains glucose, and D-pantothenic acid in a glucose and xcex2-alanine-containing nutrient medium. Furthermore, in EP-A 0 590 857 and U.S. Pat. No. 5,518,906, it is stated that the production of D-pantoic acid is improved in a glucose-containing nutrient media and the production of D-pantothenic acid is improved in a nutrient medium which contains glucose and xcex2-alanine after amplification, in the strains mentioned above, of the pantothenic acid biosynthesis genes panB, panC and panD, which should be present on the plasmid pFV3 1.
Furthermore, WO 97/10340 reports on the beneficial effect of enhancing the ilvGM operon on the production of D-pantothenic acid. Finally, EP-A-1001027 reports on the effect of enhancing the panE gene on the formation of D-pantothenic acid.
According to known procedures, D-pantothenic acid or the corresponding salt is isolated from the fermentation broth and purified (EP-A-0590857 and WO 96/33283) and then used in purified form or the entire D-pantothenic acid-containing broth is dried EP-A-1050219) and used in particular as a foodstuffs additive.
In view of the increasing demand for D-pantothenic acid, there remains a need for new methods for producing this material.
It is an object of the present invention to provide methods of making D-pantothenic acid and salts thereof, as well as feedstuffs additives containing the same.
The invention provides a process for the preparation of D-pantothenic acid and/or its salts or foodstuffs additives which contain, in addition to these, further constituents from the fermentation by fermentation of microorganisms from the Enterobacteriaceae family, in particular those which already produce D-pantothenic acid, in which
(a) at least one of the nucleotide sequences in the microorganisms coding for endogenic genes chosen from the group gcvT, gcvH and gcvP are enhanced, in particular overexpressed, under conditions which are suitable for the production of gene products (proteins),
(b) D-pantothenic acid and/or its salts are enriched in the medium or in the cells of the microorganisms and
(c) the desired products are isolated after completion of fermentation, wherein an amount of xe2x89xa70 to 100% of the biomass and/or optionally further constituents of the fermentation broth are separated,
wherein the microorganisms produce D-pantothenic acid.
The invention also provides a process in which, after completion of fermentation, all or some of the biomass remains in the fermentation broth and the broth obtained in this way is processed, optionally after being concentrated, to give a solid mixture which contains D-pantothenic acid and/or its salts and which preferably contains other constituents of the fermentation broth.
Thus, the present invention provides a method of producing D-pantothenic acid and/or a salt thereof, comprising:
fermenting a microorganism of the family Enterobacteriaceae, in which at least one of the nucleotide sequences in the microorganism coding for endogenic genes selected from the group consisting of gcvT, gcvH, and gcvP is enhanced, in a medium suitable for the production of the corresponding gene products, wherein the microorganism produces the D-pantothenic acid and/or a salt thereof.
The present invention also provides a method of producing a feedstuffs additive, comprising:
producing D-pantothenic acid and/or a salt thereof as described above, and
combining the D-pantothenic acid and/or a salt thereof with a carrier suitable for use in feedstuffs.
The present invention also provides a vector suitable for expressing one or more of the nucleotide sequences from E. coli coding for genes selected from the group consisting of gcvT, gcvH, and gcvP which contain a promoter and the one or more nucleotide sequences.
The present invention also provides a microorganism from the Enterobacteriaceae family which is transformed with the vector described above.
The present invention also provides a method for producing D-pantothenic acid and/or its salts by the fermentation of the microorganism described above.
The present invention also provides a method for producing an animal feedstuffs additive, comprising:
(a) producing D-pantothenic acid or a salt thereof as described above, wherein the alkaline earth metal of the alkaline earth salt is magnesium and/or calcium,
(b) optionally, removing water from the medium,
(c) separating the biomass formed during the fermentation in an amount of 0 to 100%,
(d) optionally, adding one or more magnesium and/or calcium salts of D-pantothenic acid to the fermentation broths from (b), and
(e) producing the feedstuffs additive,
wherein the amount of the added one or more magnesium and/or calcium salts of D-pantothenic acid is such that the amount thereof in the feedstuffs additive is in the range from about 20 to 80 wt. % based on the dry mass of the feedstuffs additive.